In essentially all self-renewing tissues, a balance is struck between cell production by mitogenesis and cell loss due to programmed cell death, otherwise known as apoptosis, thereby maintaining total cell numbers within a physiologically appropriate range. In pathological conditions, however, the balance in cell production and cell loss can be disrupted. In cancer, for example, an increased amount of cell production due to a shortened cell cycle time or a decreased amount of cell death due to dysregulation of a programmed cell death pathway results in the growth of a tumor.
Suppression of apoptosis contributes to carcinogenesis by several mechanisms, including aberrantly prolonging the cell life span, thus facilitating the accumulation of gene mutations, permitting growth factor-independent cell survival, promoting resistance to immune-based cytotoxicity, and allowing disobeyance of cell cycle checkpoints that would normally induce apoptosis. Defects in apoptotic mechanisms also play an important role in resistance to chemotherapy and radiation.
Survivin is a recently described member of the Inhibitor of Apoptosis Protein (IAP) family of antiapoptotic proteins, which are conserved across evolution with homologues found in both vertebrate and invertebrate animal species. The baculovirus IAPs, Cp-IAP and Op-IAP, were the first members of this family to be identified based on their ability to functionally complement defects in the cell death inhibitor p35, a baculovirus protein that binds to and inhibits caspase. Subsequently, five additional human (XIAP, c-IAP1, c-IAP1, cIAP2, NAIP, and Survivin) and two Drosophila homologues have been identified, which have been demonstrated to inhibit cell death. A central role for IAP-family proteins in programmed cell death regulation in Drosophila has been suggested by the finding that several apoptosis-inducing proteins in flies, including reaper, hid, and grim bind to IAPs as part of their cytotoxic mechanism. The human IAPs (XIAP, cIAP1, and cIAP2) have been reported to bind and potently inhibit caspase-3 and -7, with Kis in the range of 0.2–10 nM. These caspases operate in the distal portions of apoptotic protease cascades, functioning as effectors rather than initiators of apoptosis.
The common structural feature of all IAP family members is a ˜70 amino acid motif termed BIR, which is present in one to three copies. Using a mutagenesis approach, it was shown that the second of the three BIR domains (BIR2) of XIAP is necessary and sufficient for inhibiting certain caspase-family cell death proteases, implying that a single BIR domain can possess antiapoptotic activity. Survivin contains a single BIR domain that shares amino acid sequence similarity to the BIR2 region of XIAP.
The Survivin protein is abundantly expressed during fetal development in humans, but rarely present in adult tissues. However, expression of Survivin has been reported in most human tumors, suggesting that alterations in Survivin gene regulation occur commonly during tumorigenesis. Overexpression of Survivin in a lymphokine-dependent hematopoetic cell line has been reported to delay cell death induced by factor withdrawal. Conversely, antisense-mediated suppression of Survivin epression induces apoptosis in HcLa cells.
Most human cancers inappropriately over-express Survivin, making it one of the most tumor-specific transcripts detected to date in genome-wide surveys. Survivin has been found to be highly expressed in rapidly dividing cells but undetectable in normal differentiated tissues.
As set forth above, most IAPs bind and inhibit caspase-family cell death proteases, thus explaining their anti-apoptotic mechanism. Survivin has been reported to be associated with certain caspases, such as Caspases-3 and -7 (Tamm et al., 1998, Canc. Res., 58:5315–5320), as well as shown to block apoptosis induced by over-expression of certain caspases. Survivin also binds an IAP inhibitory protein called SMAC/Diablo and may prevent it from promoting cell death (Chunying et al. 2000, Cell, 102:33–42; Verhagen et al., 2000, Cell, 102:43–53; Chai et al., 2000, Nature, 406:855–862). However, unlike other IAPs, Survivin expression is regulated in a cell cycle-dependent manner, with maximum levels occurring during G2/M phase, and the protein localizes to mitotic spindle microtubules and midbodies of dividing cells. Therefore, Survivin is a candidate molecule that may represent an interface between apoptosis and cell cycle regulation.
In addition to apoptosis, intereference with Survivin function by antisense methods or expression of dominant-negative mutants causes polyploidy and multinuclearity, due to a cytokinesis defect. Indeed, genetic evidence has been presented that, like mammalian Survivin, its homologues Bir1p and BIR1 in yeast and C elegans, respectively, play important roles in chromosome segregation and cell division during anaphase and telophase. Moreover, Survivin physically associates with the cyclin-dependent kinase cdc2 on the mitotic apparatus, and is phosphorylated on Thr34 by cdc2/cyclin B1. Over-expression of a T34A non-phosphorylatable mutant of Survivin causes massive apoptosis and polyploidy, indicating that phosphorylation of Survivin is critical for its activity as a regulator of cytokinesis and for maintaining cell survival.
A need therefore exists to identify proteins involved in the regulation of Survivin activity for therapeutic applications, including treatment of cancer. The present invention satisfies this need and provides related advantages as well.